An interdisciplinary approach to developing tools to study antibiotic permeability in Gram-negative bacteria

Rising antibiotic resistance is an imminent threat to modern healthcare. Most new antibiotics have a narrower spectrum of activity and work only in Gram-positive bacteria. The World Health Organisation published a list of high priority bacteria with startling emerging resistance, including Gram-negative bacteria such as Pseudomonas , Klebsiell a, and Escherichia . Gram-negative bacteria are inherently more resistant to antimicrobials, due to their outer membrane, which reduces the permeability of antimicrobials. This innate resistance presents challenges in developing novel antibiotics. A high-throughput universal Gram-negative permeability assay would streamline the drug development process, enabling new broad-spectrum antibiotics to reinvigorate the drug pipeline. This thesis will explore potential avenues for monitoring and predicting permeability. The following work is an interdisciplinary investigation into Gram-negative permeability and toolkit development. This project has utilised tools from biochemistry such as enzyme assays and mass spectrometry to synthetic biology and computer science. This work approaches permeability in four ways. Firstly a potential fluorescent derivative of ampicillin was synthesised and analysed. Secondly, various proteomic techniques were utilised to monitor the covalent binding of β-lactams to penicillin-binding proteins, as a proxy of β-lactam permeability. Thirdly, bottom-up synthetic cells were created to model permeability in bacteria. Finally, machine learning algorithms consisting of supervised and unsupervised techniques were used to predict permeability. In this work alternative methods to quantifying β-lactam permeability were trialled in chapters 3 and 4. Whereas in chapters 5 and 6, the foundations to a new permeability assay and permeability predictions were laid. Ultimately, the creation of tools to predict and quantify permeability will come from many fields. Without a better understanding of permeability, antimicrobial drug development will stagnate. Therefore, to deter the impending antimicrobial resistance crisis, drug development needs to be faster, more intelligent, and better thought out; this could be achieved with a permeability assay used in conjunction with activity screens